Process for the synthesis of modified P-chiral nucleotide analogues

ABSTRACT

The process of the instant invention is drawn to the synthesis of modified P-chiral nucleotide analogues in the form of pure diastereomers possessing preselected configuration at the P-atom. Oligonucleotides prepared by the method of the invention containing P-chiral compounds have enhanced hybridization and transporting properties.

An object of the invention is to provide a process for the synthesis ofmodified P-chiral nucleotide analogues of general formula 1, where R₁stands for protecting group, preferably 4,4′-dimethoxytrityl (DMT),9-phenylxanthene-9-ol (Px) or trialkylsilyl group, R₂ is a hydrogenatom, protected hydroxyl group, halogen, chloroalkyl, nitrile, azide,protected amine, perfluoroalkyl (containing up to four carbon atoms),perfluoroalkoxyl (containing up to four carbon atoms and up to ninefluorine or chlorine atoms), alkoxyalkyl, vinyl, ethynyl, OQ₁, SQ₁,NHQ₁, where Q₁ stands for alkyl (C₁-C₄), aryl (C₆-C₁₂), alkenyl (C₃-C₁₂)or alkynyl (C₃-C₁₂), B stands for a purine or pyrimidine base(appropriately protected if necessary), Z is selected from Q₁ or vinyl,ethynyl, aminomethyl or aminoethyl substituents, X means oxygen, sulfuror selenium atom, R_(x) is a protecting group, preferably aroyl, acyl,alkoxycarbonyl, benzenesulfonic, alkyl, trialkylsilyl group or the nextunit of elongated oligonucleotide chain.

Bacterial or viral infection, as well as uncontrolled proliferation ofcancer cells in a living organism, lead to a fully developed diseasepredominantly by synthesis of “unwanted”, harmful proteins. Viraldiseases result from incorporation of viral genetic information into ahost's genome followed by synthesis of viral proteins, which aredamaging to the host organism.

Caused by different factors aberrations of protooncogenes and formationof oncogenes responsible for synthesis of “unwanted” proteins arerecognized as important factors in cancer cells proliferation processes.

Recent achievements in molecular biology, including explanation ofmolecular bases of such diseases as AIDS, different viral and cancerdiseases or blood circulation disesaes, resulted in intensive search fornew selective treatments aimed at inhibition of the expression of geneswhich code “unwanted” proteins, or at tuning of the level of knownregulatory proteins.

Two newly developed therapeutic approaches are ANTISENSE mRNA (C. A.Stein, Cancer Res., 1988, 48, 2659) and ANTIGENE (N. T. Thuong et al.,Angew.Chem.Int.Ed.Engl., 1993, 32) strategies, which stem from theknowledge on interactions between oligo(deoxyribonucleotide)s and DNA orRNA molecules. These conceptions are based on the assumption that shortsynthetic oligo(deoxyribonucleotide)s after being delivered inside acell, form stable duplexes with complementary DNA or RNA molecules, andon this way slow down either transcription or translation process (E.Wickstrom, ed. Wiley-Liss, New York N.Y. 1993, “Prospects for AntisenseNucleic Acid Therapy for Cancer and AIDS”).

Nucleolytic enzymes present in cells and body fluids are able tohydrolyze exogenous DNA molecules very rapidly, thus stability ofoligo(deoxyribonucleotide)s and their analogues against nucleases is acrucial factor in respect to their in vivo activity. Majority ofmodifications introduced to the oligo(deoxyribonucleotide)s with the aimof their enhanced nucleolytic stability, involved changes of ligandsattached to the phosphorus atom of the internucleotide phosphodiesterbond. Among them phosphorothioate, methanephosphonate, phosphoramidateand triester analogues to various extent fulfill the criterion of fullor, at least, significantly enhanced stability. However, suchmodifications usually result in reduced hybridization properties towardscomplementary DNA and RNA strands (J. S. Cohen, ed. Oligonucleotides:Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton,Fla., 1989).

Applicability of antisense oligonucleotides as potential therapeuticsdepends upon their ability to cross the cellular membranes to reachnecessary therapeutic concentration at the site of target moleculesinside the cell (e.g. mRNA in cytoplasm). The cellular membranes made ofprotein-lipid layers are permeable only for small non-ionic moleculesand are not permeable for most of natural metabolites and many drugs.

Natural and modified oligonucleotides complementary to fragments ofviral DNA (RNA) are reported to show antiviral and anticancer propertiesin cell lines (in vivo), thus they are able to permeate through cellmembranes and hybridize to the target DNA or RNA molecules. Severalnucleolytically stable DNA analogues, as alkyl triesters (P. S. Miller,Biochemistry, 1977, 16, 1988), and methanephosphonates (C. H.Marcus-Sekura et al., Nucleic Acids Res., 1987, 15, 5749; P. S. Milleret al., Biochemistry, 1986, 25, 5092; S. K. Loke et al., Top. Microbiol.Immunol., 1988, 141, 282; A. M. Tari et al., J.Biol.Med., 1996, 74, 623;S. Agrawal et al., Clin.Pharmacokinet., 1995, 28, 7) were used for theresearch in different cell lines including human HL60, Syrian hamsterfibroblasts, U 937, L 929, CV-1 and ATH 8. For modified oligonucleotidesthe cellular uptake is usually rather low, what results in reduced invivo activity compared to that expected from in vitro studies.

So far, DNA analogues have worse hybridization properties than naturalDNA, thus the inhibition of transcription or translation, and,consequently, inhibition of protein biosynthesis are less effective thanexpected. There are several reasons for this phenomenon, such ascomplicated third-order structure of RNA, limited accessibility of itsparticular segments, or DNA/RNA interactions with proteins.

In order to overcome these obstacles several DNA analogues possessinginternucleotide linkages without phosphorus atom, like methylene group(M. Matteuci, Tetrahedron Lett., 1990, 31, 2385) dialkylsilyl groups (R.Stirczak, J.Org.Chem., 1987, 52, 202) or sulfonyl group (S. Benner,J.Org.Chem., 1995, 61, 7620) have been synthesized. Research on theirapplication as therapeutics is in an initial phase, mostly because ofunfavorable physicochemical properties, as poor solubility andhybridization properties, and low chemical stability. Triester analoguesare degradable by esterases, what renders them unusable in the antisensestrategy (Goodrick et al., Bioconj.Chem., 1990, 1, 165).

In the case of phosphorothioate and methanephosphonate analogues of DNA,which possess chiral center at the phosphorus atom, an additionalproblem is encountered, since the synthesis of oligomers with ninternucleotide bonds results in formation of 2^(n) diastereoisomers,unless the method of synthesis is stereospecific.

It was found, that for oligo(nucleoside-3′,5′-methanephosphonate)s ofR_(P)-, S_(P)- or random configuration at each phosphorus atom, theirhybridization properties towards complementary DNA or RNA depend on theconfiguration of the phosphorus centers (P. S. Miller et al.,J.Biol.Chem., 1980, 255, 9659; Biochemistry, 1982, 21, 2507). Forphosphorothioate DNA analogues the stereodifferentiation ofhybridization properties is accompanied by their stereoselectivesusceptibility to enzymatic hydrolysis by certain nucleases (Potter etal., Biochemistry, 1983, 22, 1369; Bryant et al., Biochemistry, 1979,18, 2825).

Leśnikowski et al.(Nucleic Acids Res., 1990, 18, 2109) found thatstereospecifically synthesized octamer possessing six out of seveninternucleotide methanephosphonate bonds of R_(P) configuration has muchstronger affinity towards pentadeoxyadenylic template than itscounterpart possessing these bonds of S_(P) configuration, or theoligomer obtained by non stereoselective method. The stereoregularoligomers were obtained by non stereoselective condensation ofcorresponding two stereoregular tetramers synthesized in solutionstarting from diastereomerically pure5′-O-MMT-thymidine-3′-O-(O-p-nitrophenylmethanephosphonate)s and3′-O-acetylthymidine with Grignard reagent used as an activator(Leśnikowski et al., Nucleic Acids Res., 1990, 18, 2109; ibid, 1988, 16,11675; Leśnikowski et al., Nucleosides & Nucleotides, 1991, 10, 773).

Other examples of synthesis of diastereomerically pure (or, at least,significantly enriched with an R_(P) diastereoisomer) methanephosphonateanalogues of DNA involve reactions of methyidichlorophosphine withappropriately protected at the 5′ (first step) and 3′ (second step)nucleosides, carried out at low temperature (−80° C.) in the presence ofamines (including chiral amines). The highest obtained ratio of R_(P) toS_(P) isomers was 8:1 (Loscher, Tetrahedron Lett., 1989, 30, 5587;Engels et al., Nucleosides & Nucleotides, 1991, 10, 347) This methodallows to synthesize dinucleoside methanephosphonates indiastereoselective manner.

Another method for stereoselective formation of internucleotidemethanephosphonate bond is a reaction employing separateddiastereoisomers of 5′-O-DMT-N-protected nucleoside3′-O-(Se-alkylmethanephosphonate)s and appropriate3′-5′-OH-(N-protected) nucleosides in the presence of DBU and lithiumchloride (Woźniak et al.,J.Org.Chem., 1994, 58, 5061).

Recently, numerous laboratories have paid efforts to implement astherapeutics so called “chimeric” oligomers, possessing phosphate orphosphorothioate “core” flanked at both 5′ and 3′ ends bymethanephosphonate units of R_(P) configuration. The chimeras haveenhanced stability against nucleases due to the presence ofenzymatically stable internucleotide methanephosphonate linkages.Incorporation of methanephosphonate units only of R_(P) configurationresults in enhanced hybridization properties of the “chimeric” product(M. Reynolds et al., Nucleic Acids Res., 1996, 24, 4584).

A process for the synthesis of modified P-chiral nucleotide analogues ofgeneral formula 1, where:

R₁ stands for protecting group, preferably 4,4′-dimethoxytrityl (DMT),9-phenylxanthene-9-ol (Px) or trialkylsilyl group,

R₂ is a hydrogen atom, protected hydroxyl group, halogen, chloroalkyl,nitrile, azide, protected amine, perfluoroalkyl (containing up to fourcarbon atoms), perfluoroalkoxyl (containing up to four carbon atoms andup to nine fluorine or chlorine atoms), alkoxyalkyl, vinyl, ethynyl,OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl (C₁-C₄), aryl (C₆-C₁₂),alkenyl (C₃-C₁₂) or alkynyl (C₃-C₁₂),

B stands for a purine or pyrimidine base (appropriately protected ifnecessary),

Z is selected from Q₁ or vinyl, ethynyl, aminomethyl or aminoethylsubstituents,

X means oxygen, sulfur or selenium atom,

and R_(x) is a protecting group, preferably aroyl, acyl, alkoxycarbonyl,benzenesulfonic, alkyl, trialkylsilyl group or the next unit ofelongated oligonucleotide chain

according to the present invention, consists in reaction of compound offormula 2, where R₁, R₂, B and Z have the above mentioned meanings,while Y stands for XR₃ substituent, where X means oxygen, sulfur orselenium atom, and R₃ means acyl group of formula COR₄, in which R₄stands for alkyl (up to six carbon atoms), perfluoroalkyl (containing upto four carbon atoms), aroyl (containing six up to fifteen carbonatoms), preferably mono-, di- or trisubstituted aromatic substituents(—C₆H₄R₅, —C₆H₃(R₅)₂ or—C₆H₂(R₅)₃, respectively), where R₅ means ahydrogen atom, methyl substituent, halogen atom or other substituentactivating the aromatic ring, with compound of formula 6, where B, R₂and R_(x) have the above mentioned meanings, under anhydrous conditions,in an aprotic organic solvent, in the presence of an activating reagent,to yield compound of formula 1, which then is isolated, and if X means asulfur or selenium atom compound of formula 1 is oxidized with knownoxidizing reagents, preferably a mixture iodine/water/pyridine, hydrogenperoxide, alkyl hydroperoxides (preferably t-butyl hydroperoxide), orpotassium peroxymonosulfate, followed by isolation of resulting 1 (whereX means an oxygen atom and R₁, R₂, R_(x), B and Z have the abovementioned meanings) using known methods. The process according to thepresent invention is carried out preferably in tetrahydrofuran oracetonitrile.

As an activating reagent in the reaction between compounds of formula 2and formula 6 one can use organic bases, preferably amines, morepreferably 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or1,5-diazabicyclo[4.3.0]non-5-ene (DBN).

In the process according to the present invention it is preferred to usean additional activator selected from a group consisting of lithiumsalts, especially lithium halides.

Another variant of the process for the synthesis of modified P-chiralnucleotide analogues of general formula 1 in the form of purediastereomer of preselected configuration at the P-atom, where R₁, R₂,R_(x), B, X and Z have the above mentioned meanings, according to thepresent invention consist in reaction of one of two diastereomers ofcompound of formula 2 of the configuration at the P-atom identical tothat desired in the product, where R₁, R₂, B and X have the abovementioned meanings, while

Y stands for XR₃ substituent, where X means an oxygen, sulfur orselenium atom, R₃ means acyl group of formula COR₄, in which R₄ standsfor alkyl (up to six carbon atoms), perfluoroalkyl (containing up tofour carbon atoms), aryl (containing six up to fifteen carbon atoms),including mono-, di- or tri-substituted aromatic substituents (—C₆H₄R₅,—C₆H₃(R₅)₂ or —C₆H₂(R₅)₃, respectively), where R₅ means a hydrogen atom,methyl substituent, halogen atom or other substituent activating thearomatic ring,

with compound of formula 6, where B, R₂ and R_(x) have the abovementioned meanings, under anhydrous conditions, in an aprotic organicsolvent, in the presence of an activating reagent, to yield compound offormula 1, which then is isolated, or, if X means a sulfur or seleniumatom, compound of formula 1 is oxidized with known oxidizing reagents,preferably a mixture iodine/water/pyridine, hydrogen peroxide, alkylhydroperoxides (preferably t-butyl hydroperoxide), or potassiumperoxymonosulfate, followed by isolation of resulting 1 (where X meansan oxygen atom and R₁, R₂,R_(x), B and Z have the above mentionedmeanings) using known methods.

The process according to the present invention is carried out preferablyin tetrahydrofuran or acetonitrile. As the activating reagent in thereaction between compounds of formula 2 and formula 6 one can useorganic bases, preferably amines, more preferably1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or1,5-diazabicyclo[4.3.0]non-5-ene (DBN), and as an additional activatorcompounds selected from a group consisting of lithium salts, especiallylithium halides, can be used.

The third variant of the process for the synthesis of modified P-chiralnucleotide analogues of general formula 1 in the form of purediastereomer of preselected configuration at the P-atom, where R₁, R₂,R_(x), B, X and Z have the above mentioned meanings, according to thepresent invention consist in hydrolysis of one of two diastereomers ofcompound of formula 2 of the configuration at the P-atom opposite tothat desired in the product of formula 1, while in the formula 2 R₁, R₂,B, Z, X and Y have the above mentioned meanings, in the presence ofactivator being able to invert an absolute configuration of the P-atom,while resulting product of general formula 2, where R₁, R₂, and B havethe above mentioned meanings, while Y stands for an oxygen atom and Xmeans a sulfur or selenium atom, is reacted with compound of generalformula 7, where R₄ stands for alkyl (up to six carbon atoms),perfluoroalkyl (containing up to four carbon atoms), aroyl (containingsix up to fifteen carbon atoms), including mono-, di- or tri-substitutedaromatic substituents (—C₆H₄R₅, —C₆H₃(R₅)₂ or —C₆H₂(R₅)₃, respectively),where R₅ means a hydrogen atom, methyl substituent, halogen atom or anyother substituent, and W means a chlorine, bromine or iodine atom, toyield compound of formula 2, where R₁, R₂, B and Z have the abovementioned meanings, X means a sulfur or selenium atom, while Y standsfor R₄C(O)O—, in which R₄ has the above mentioned meaning, possessingthe absolute configuration at the P-atom opposite to that in thestarting material, and identical to that required for the product offormula 1, further possibly combined with the same diastereoisomer offormula 2 obtained from the earlier separation, and then reacted withcompound of formula 6, where B, R₂ and R_(x) have the above mentionedmeanings, under anhydrous conditions, in an aprotic organic solvent, inthe presence of an activating reagent, to yield compound of formula 1 ofdesired absolute configuration at the P-atom, which then is isolated,or, if X means a sulfur or selenium atom, compound of formula 1 isoxidized with known oxidizing reagents, preferably a mixtureiodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides(preferably t-butyl hydroperoxide), or potassium peroxymonosulfate,followed by isolation of resulting 1 (where X means an oxygen atom andR₁, R₂, R_(x), B and Z have the above mentioned meanings) using knownmethods. The process according to the present invention is carried outpreferably in tetrahydrofuran or acetonitrile.

As an activating reagent in the reaction between compounds of formula 2and formula 6 one can use organic bases, preferably amines, morepreferably 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and as an additional activatorlithium salts, especially lithium halides, are used.

The fourth variant of the process for the synthesis of modified P-chiralnucleotide analogues of general formula 1 in the form of purediastereomer of preselected configuration at the P-atom, where R₁, R₂,R_(x), B and Z have the above mentioned meanings, according to thepresent invention consist in reaction of one of two diastereomers offormula 2 of the configuration at the P-atom opposite to that desired inthe product 1, while in the formula 2 R₁, R₂, B, Z, X and Y have theabove mentioned meanings, with alcohol, preferably with methanol,possibly in the presence of activator, while the resulting product ofgeneral formula 2, where R₁, R₂, Z and B have the above mentionedmeanings, while Y stands for an alkoxyl group, preferably methoxyl, andX means a sulfur or selenium atom, is further dealkylated using amines,preferably trimethylamine or t-butylamine, and the resulting compound offormula 2, where R₁, R₂, Z and B have the above mentioned meanings,while Y stands for an oxygen atom and X means a sulfur or selenium atom,is subsequently reacted with compound of general formula 7, where R₄ andW have the above mentioned meanings, to yield compound of formula 2,where R₁, R₂, Z and B have the above mentioned meanings, X means asulfur or selenium atom, while Y stands for R₄C(O)O—, possessing theabsolute configuration at the P-atom opposite to that in the startingmaterial, and identical to that required for the product of formula 1,further possibly combined with the same diastereoisomer of formula 2obtained from the earlier separation, and then reacted with compound offormula 6, where B, R₂ and R_(x) have the above mentioned meanings,under anhydrous conditions, in an aprotic organic solvent, in thepresence of an activating reagent, to yield compound of formula 1 ofdesired absolute configuration at the P-atom, which then is isolated,or, if X means a sulfur or selenium atom, compound of formula 1 isoxidized with known oxidizing reagents, preferably a mixtureiodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides(preferably t-butyl hydroperoxide), or potassium peroxymonosulfate,followed by isolation of resulting 1 (where X means an oxygen atom andR₁, R₂, R_(x), B and Z have the above mentioned meanings) using knownmethods. The process according to the present invention is carried outpreferably in tetrahydrofuran or acetonitrile.

As an activating reagent in the solvolysis and in the reaction betweencompounds of formula 2 and formula 6 one can use organic bases,preferably amines, more preferably 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) or 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and as an additionalactivator lithium salts, especially lithium halides, are used.

In the process according to the present invention preferably usedcompounds are those of general formula 2, obtained by phosphorylation ofcorresponding substrates of general formula 3, where R₁, R₂ and B havethe above mentioned meanings, with phosphorylating reagents of generalformula 4, where Z and X have the above mentioned meanings, W means ahalogen atom, preferably chlorine, followed by hydrolysis withoutisolation of the intermediate 5, to yield compounds of formula 2, whereR₁, R₂, B, Z and X have the above mentioned meanings, and Y means anoxygen atom.

Using the first variant of the process according to the presentinvention, pure diastereoisomers of formula 2 are transformed separatelyto yield pure diastereoisomers of compound 1.

More useful variant of the process according to the present inventionconsist in the reaction of compound of formula 3, where R₁ and B havethe above mentioned meanings, with compound of formula 4, where X meansa sulfur or selenium atom and Z has above mentioned meanings, to yieldcompound of formula 5, where X means an oxygen, sulfur or selenium atom,which is then hydrolyzed to yield compound of formula 2 where R₁, R₂, Zand X have the above mentioned meanings and Y means an oxygen atom, andseparated chromatographically into two diastereomers, followed byreaction with compound of formula 7, where R₄ stands for alkyl (up tosix carbon atoms), perfluoroalkyl (containing up to four carbon atoms),aroyl (containing six up to fifteen carbon atoms), including mono-, di-or tri-substituted aromatic substituents (—C₆H₄R₅, —C₆H₃(R₅)₂ or—C₆H₂(R₅)₃, respectively), where R₅ means a hydrogen atom, methylsubstituent, halogen atom or any other substituent activating anaromatic ring, and W means a halogen, preferably chlorine. One isomer isreacted with compound of formula 6, to yield stereospecifically compoundof formula 2, where Y stands for R₄C(O)O—, in which R₄ has the abovementioned meaning. This isomer of 2 is reacted with compound of formula6, where B, R₂ and R_(x) have the above mentioned meanings, in thepresence of an activating reagent as an organic base, preferablytertiary amine, more preferably 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU). Diastereomerically pure compound of formula 1 is isolated usingknown methods. In the process according to the present invention theproduct 1 is oxidized with known oxidizing reagents, preferably amixture iodine/water/pyridine, hydrogen peroxide, alkyl hydroperoxides(preferably t-butyl hydroperoxide), or potassium peroxymonosulfate, toyield product 1, where X means an oxygen atom and R₁, R₂, R_(x), B and Zhave the above mentioned meanings.

In this variant the second diastereoisomer of 2 (Y=R₄C(O)O—) is reactedwith alcohol (preferably methanol), and without isolation ofintermediary 2, where R₁, R₂, Z and B have the above mentioned meanings,while Y stands for an alkoxyl group, preferably methoxyl, and X means asulfur or selenium atom, is further dealkylated using strong base,preferably organic base, most preferably amine. This diastereoisomer hasan absolute configuration opposite to that of the substrate 2, thuswithin the described above process inversion of configuration at theP-atom in compound of formula 2 takes place. The described variant ofthe invention allows to obtain compound of formula 2 (Z=O, X=S, Se) inwhich absolute configuration at the phosphorus atom is 100% inverted,starting from 2 (Y=R₄C(O)O—) without isolation of intermediary 2 (Z=OMe,X=S, Se). It allows also to use both separated diastereomers of 2 (Y=O,X=S, Se) for synthesis of one diastereomer of the same compound offormula 2 of desired configuration at the P-atom via compound of formula2 (X=S or Se, Y=R₄C(O)O—).

Within the next variant of the invention, one of the separateddiastereomers of 2 (Y=O, X=S, Se) possessing an absolute configurationidentical with that desired for the product 1, is alkylated with knownalkylating reagents, preferably alkyl halides 8 of general formula R₆W,where R₆ stands for methyl, cyanomethyl, halogenoacyl, benzyl oraromatic ring substituted benzyl, while W means a chlorine, bromine oriodine atom. The resulting compound of formula 2, where

a) R₁ stands for protecting group, preferably 4,4′-dimethoxytrityl(DMT), 9-phenylxanthene-9-ol (Px) or trialkylsilyl group,

b) R₂ is a hydrogen atom, protected hydroxyl group, halogen,chloroalkyl, nitrile, azide, protected amine, perfluoroalkyl (containingup to four carbon atoms), perfluoroalkoxyl (containing up to four carbonatoms and up to nine fluorine or chlorine atoms), alkoxyalkyl, vinyl,ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl (C₁-C₄), aryl(C₆-C₁₂), alkenyl (C₃-C₁₂) or alkynyl (C₃-C₁₂),

c) B stands for a purine or pyrimidine base (appropriately protected ifnecessary),

d) Z is selected from Q₁ or vinyl, ethynyl, aminomethyl or aminoethylsubstituents,

X means oxygen and Y means SR₆ or SeR₆, where R₆ has the above mentionedmeaning, is reacted with compound of formula 6, where B stands for apurine or pyrimidine base (appropriately protected if necessary), andR_(x) is a protecting group, preferably aroyl, acyl, alkoxycarbonyl, orthe next unit of elongated oligonucleotide chain. This reaction iscatalyzed by strong bases, preferably organic bases as1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). An additional activator ofthis process may be selected from a group consisting of lithium salts,preferably lithium halides, most preferably lithium chloride. Theresulting compound of formula 1, where X means an oxygen atom and othersubstituents have the above mentioned meanings, is isolated using knownmethods. This product has the absolute configuration identical to thatof the product 1 obtained by oxidation of compound of formula 1, where Xmeans a sulfur or selenium atom.

The second diastereomer of of formula 2 (X=S, Se, Y=O) is acylated withcompound of formula of formula 7, and then condensed with compound offormula 6 as in the second variant of the process.

The resulting compound of formula 1, where X means a sulfur or seleniumatom is isolated using known methods, and oxidized using known oxidizingreagents, preferably a mixture iodine/water/pyridine, hydrogen peroxide,alkyl hydroperoxides (preferably t-butyl hydroperoxide), or potassiumperoxymonosulfate, to yield compound 1, where X means an oxygen atom andR₁, R₂, R_(x), B and Z have the above mentioned meanings.

This means, that described above variant of the invention allows,starting from both separated diastereoisomers of 2 (X=S, Se, Y=O) whichare independently converted on two different ways (vide supra) to yieldone diastereoisomer of the product 1 of desired absolute configurationat the P-atom, where X means an oxygen atom and other substituents havethe above mentioned meanings.

The examples of the process according to the invention, not limiting itsscope, are presented below.

EXAMPLES 1-8

General method for synthesis of compounds of formula 2 (Z=Me, X=S or Se,Y=O).

To the solution of compound of general formula 3 (1 mmol) in pyridine,compound of general formula 4 (Z=Me, X=S or Se) was added, and thereacting mixture was stirred for 15 min. Then water was added and thestirring was continued for 10 minutes. The mixture was evaporated todryness under reduced pressure. The residue was dissolved in chloroform,washed twice with NaHCO₃aq. The organic layer was dried with knowndrying agents (e.g. magnesium sulfate) and concentrated under reducedpressure. The resulting crude product was purified and/or separated intodiastereomeric species by means of column chromatography.

Appropriate fractions were collected and evaporated to yield colorlessfoam, and finally precipitated from a mixture of chloroform (ormethylene chloride) and petroleum ether.

Selected experimental details are collected in Table 1

TABLE 1 Examples B R_(z) ³¹P NMR* (ppm) Yield (%) 1 T H 77.67; 77.34 922 ^(Bz)A H 78.33; 78.77 90 3 ^(Bz)C H 76.41; 77.00 93 4 ^(ibu)G H 78.28;78.97 85 5 U OMe 78.24; 77.96 90 6 A^(Bz) OMe 78.24; 78.39 83 7 C^(Bz)OMe 78.83; 79.54 85 8 G^(ibu) OMe 79.03; 79.15 80 *in CDCl₃, aspyridinium salts

EXAMPLES 9-17

General method for synthesis of compounds of formula 2 (Z=Me, X=S or Se,Y=O(CO)R₄).

To the solution of 1 mmol of compound of formula 2 (X=S, Y=O) inpyridine (5 mL) compound of formula 7 was added (2-3 mmol) and thereacting mixture was stirred at room temperature until the substratedisappeared (TLC control). The mixture was concentrated under reducedpressure and oil residue was dissolved in chloroform. Purification wasdone either by column chromatography or precipitation from a mixturechloroform/petroleum ether.

Selected experimental details are collected in Table 2.

TABLE 2 Yield Nr B R_(z) R₄ X ³¹P NMR (%)* 9 T H 2,4,6-trimetylphenyl S91.6 +98 10 A^(Bz) H ″ S 91.3 +98 11 C^(Bz) H ″ S 91.8; 91.3 +98 12G^(ibu) H ″ S 91.8; 92.3 96 13 U OMe ″ S +98 14 A^(Bz) H ″ Se 92.04**;91.79 +98 15 C^(Bz) H ″ Se 92.11***; 91.73 +98 16 T H2,4,6-trichlorphenyl S 93.03; 93.12 +98 17 T H phenyl S 92.12; 92.25 +98*Yield assessed from ³¹P NMR **J_(P-Se) = 916 Hz ***J_(P-Se) = 912 Hz

General method for synthesis of compounds of formula 2 (Z=Me, X=S or Se,Y=O, O-alkyl (methyl, ethyl))

To the solution of 2 (Z=Me, X=S or Se, Y=O(CO)R₄) (1 mmol) in dryacetonitrile, 5 mmol of DBU and 10-20 mmol of alcohol were added. Afterthe reaction was complete, the reaction mixture was concentrated underreduced pressure to ⅓ of initial volume, diluted with chloroform andwashed with water and NaHCO₃aq. The organic layer was dried, thesolvents were evaporated under reduced pressure and the product wasisolated by column chromatography on silica gel.

Conversion of compounds of formula 2 (Z=Me, X=S or Se, Y=O(CO)R₄) intocompounds of formula 1 (Z=Me, X=S or Se).

The reacting mixture consisting of compound of formula 2 (Z=Me, X=S orSe, Y=O(CO)R₄) (1 mmol), compound 6 (5 mmol) and DBU (20 mmol) inanhydrous acetonitrile was stirred at room temperature in an atmosphereof inert gas for 24 h. The mixture was concentrated under reducedpressure, to the residue chloroform was added and the solution wasextracted twice with 0.05 m solution of citric acid. The organic layerwas dried with magnesium sulfate, concentrated and the product wasisolated chromatographically on a silica gel column.

EXAMPLE 18

Substrate 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OC(O)C₆H₂(CH₃)₃];

FAST-[S_(P)]: ³¹P NMR d: 91.6 ppm.

Product 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OMe]

[S_(P)]: ³¹P NMR d: 100.3 ppm; diast.purity +99%; yield 92%

EXAMPLE 19

Substrate 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OC(O)C₆H₂(CH₃)₃];

SLOW-[R_(P)]: ³¹P NMR d: 91.3 ppm.

Product 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OMe]

[R_(P)): ³¹P NMR d: 99.6 ppm; diast.purity +99%; yield 92%

EXAMPLE 20

Substrate 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OC(O)C₆H₂(CH₃)₃];

FAST-[S_(P)]: ³¹P NMR d: 91.6 ppm.

Product 2:

[Y=OEt] [S_(P)]: ³¹P NMR d: 101.3 ppm; diast.purity 92%; yield 95%

EXAMPLE 21

Substrate 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OC(O)C₆H₂(CH₃)₃];

FAST-[S_(P)]: ³¹P NMR d: 91.6 ppm;

alcohol: NCCH₂CH₂OH

time: 12 hours.

Product 2:

[Y=O] [S_(P)]: ³¹P NMR d: 75.7 ppm; diast.purity 95%; yield 99%

EXAMPLE 22

Substrate 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OC(O)C₆H₂(CH₃)₃];

FAST-[S_(P)]; ³¹P NMR d: 91.6 ppm;

reaction with water; analogous reaction conditions

Product 2:

[Y=O] [S_(P)]: ³¹P NMR d: 74.3 ppm; diast.purity 100%; yield 99%

EXAMPLE 23

Substrate 2:

[R₁=DMT, B=Thy, R_(Z)=H, Z=Me, X=S, Y=OC(O)C₆H₂(CH₃)₃];

SLOW-[R_(P)]; ³¹P NMR d: 91.3 ppm;

reaction with water, analogous reaction conditions.

Product 2:

[Y=O] [R_(P)]: ³¹P NMR d: 74.65 ppm; diast. Purity 100%; yield 99%

EXAMPLE 24

Using the substrate 2 (R₁=DMT, R₂=H, B=Thy,Y=OC(O)C₆H₂(CH₃)₃-SLOW-[R_(P)] (d ³¹P NMR 91.3 ppm, diastereomericpurity 99+%), and compound 6 (R_(x)=t-BuMe₂Si, R₂=H, B=Thy) the product1 [Z=Me, X=S] FAST-[R_(P)] was obtained in 80% yield and ofdiastereomeric purity 91%, d³¹P NMR 99.3 ppm.

EXAMPLE 25

Using the substrate 2 (R₁=DMT, R₂=H, B=Thy,Y=OC(O)C₆H₂(CH₃)₃-FAST-[S_(P)] (d ³¹P NMR 91.6 ppm), and compound 6(R_(x)=t-BuMe₂Si, R₂=H, B=Thy) the product 1 [Z=Me, X=S] SLOW-[S_(P)]was obtained in 82% yield and of diastereomeric purity 90%, d³¹P NMR100.2 ppm.

General method for inversion of absolute configuration at the P-atom incompound of formula 2 [Z=Me, X=S or Se, Y=OC(O)R₄.

A substrate 2 [R₁=DMT, X=S or Se, Y=OC(O)R₄]-FAST-[S_(P)]] was dissolvedin acetonitrile and methanol (3:1 v:v) containing DBU (20 fold excess)and the solution was stirred at room temperature for 4 h. The product 2[R₁=DMT, X=S or Se, Y=OMe] after extraction and drying was reacted withcompound 7 (R₄C(O)W), and the resulting product 2 [R₁=DMT, X=S or Se,Y=OC(O)R₄]-SLOW-[R_(P)]] was isolated and purified as described inexamples 9-17.

EXAMPLE 26

Using the substrate 2 (R₁=DMT, B=Thy, X=S,Y=OC(O)C₆H₂(CH₃)₃-FAST-[S_(P)] (d³¹P NMR 91.6 ppm), the product 2(R₁=DMT, B=Thy, X=S, Y=OC(O)C₆H₂(CH₃)₃-SLOW-[R_(P)]) was obtained in 86%yield as assessed by ³¹P NMR (d ³¹P NMR 91.3 ppm).

EXAMPLE 27

Using the substrate 2 (R₁=DMT, B=Thy, X=S,Y=OC(O)C₆H₂(CH₃)₃-SLOW-[R_(P)] (d ³¹P NMR 91.3 ppm), the product 2(R₁=DMT, B=Thy, X=S, Y=OC(O)C₆H₂(CH₃)₃-FAST-[S_(P)]) was obtained in 80%yield as assessed by ³¹P NMR (d ³¹P NMR 91.6 ppm).

What is claimed is:
 1. A process for the synthesis of modified P-chiralnucleotide analogues of Formula 1:

wherein: R₁ is a protecting group; R₂ is selected from hydrogen,protected hydroxyl group, vinyl, halogen, nitrile, azide, protectedamine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl,ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenyl oralkynyl; B is a purine or pyrimidine base; Z is selected from alkyl,aryl, alkenyl, alkynyl, vinyl, ethynyl, aminomethyl or aminoethylsubstituents; X is an oxygen, sulfur or selenium atom; R_(x) is aprotecting group; said process comprising the steps of: (a) providingthe following compound of Formula 2:

wherein R₁, R₂ and B are as defined above; Y is an XR₃ substituent,wherein X is an oxygen, sulfur or selenium atom and R₃ is an acyl groupof formula COR₄, in which R₄ is an alkyl group, perfluoroalkyl group, oran aryl substituent containing six to fifteen carbon atoms and having anaromatic ring comprising mono-, di- or tri-substituted aromaticsubstituents activating the aromatic ring; (b) providing the followingcompound of Formula 6:

wherein: B, R₂ and R_(x) are as defined above; (c) reacting the compoundof Formula 6 with the compound of Formula 2 under anhydrous conditions,in an aprotic organic solvent, in the presence of at least oneactivating reagent, to yield the compound of Formula 1; and (d)isolating the compound of Formula
 1. 2. The process according to claim1, wherein R_(x) is selected from aroyl, acyl, alkoxycarbonyl,benzenesulfonic, alkyl, trialkylsilyl group and an elongatedoligonucleotide chain.
 3. The process according to claim 1, wherein X isa sulfur or selenium atom and the process comprises oxidizing thecompound of formula 1 with an oxidizing reagent, prior to the step ofisolating the compound of Formula
 1. 4. The process according to claim1, wherein X is an oxygen atom.
 5. The process according to claim 1,wherein the compound of Formula 2 is obtained by: (i) phosphorylatingwith a phosphorylating reagent, a substrate of Formula 3:

wherein R₁, R₂ and B are as defined previously, said phosphorylatingreagent comprising a compound of Formula 4:

wherein: Z is as defined previously, and W is a halogen; and (ii)followed by hydrolysis without isolation of the intermediate of Formula5:

where R₁, R₂, B, Z and X are as defined in claim 1, and Y is an oxygenatom, to yield the compound of Formula
 2. 6. The process according toclaim 1, wherein the aprotic organic solvent comprises tetrahydrofuranor acetonitrile.
 7. The process according to claim 1, wherein the atleast one activating reagent is an organic base amine.
 8. The processaccording to claim 1, wherein the reacting in step (c) comprises anadditional activating reagent which is a lithium salt.
 9. The processaccording to claim 1, wherein the compound of Formula 2 is adiastereoisomer which possesses an absolute configuration at the P-atomidentical with that desired the analogues of Formula 1, saiddiastereoisomer being reacted with the compound of Formula 6 underanhydrous conditions, in the aprotic organic solvent, in the presence ofthe at least one activating reagent, to yield the compound of Formula 1.10. The process according to claim 9, wherein the compound of Formula 2is obtained by phosphorylation of a substrate of Formula 3:

with a phosphorylating reagent of Formula 4:

followed by hydrolysis without isolation of an intermediate of Formula5:

to yield a compound of Formula 2 wherein R₁, R₂, B, Z and X are asdefined previously, and Y is an oxygen atom.
 11. The process accordingto claim 9, wherein the aprotic organic solvent is tetrahydrofaran oracetonitrile.
 12. The process according to claim 9, wherein the at leastone activating reagent is an organic base.
 13. The process according toclaim 9, wherein the reacting in step (c) is in the presence of anadditional activating reagent which is a lithium salt.
 14. A process forthe synthesis of modified P-chiral nucleotide analogues of Formula 1 inthe form of a pure diastereoisomer possessing a preselectedconfiguration at the P-atom:

wherein: R₁ is a protecting group; R₂ is selected from hydrogen,protected hydroxyl group, vinyl, halogen, nitrile, azide, protectedamine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl,vinyl, ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenylor alkynyl; B is a purine or pyrimidine base; Z is selected from alkyl,aryl, alkenyl, alkynyl, vinyl, ethynyl, aminomethyl or aminoethylsubstituents; R_(x) is a protecting group; Y is an XR₃ substituent,where X is an oxygen, sulfur or selenium atom and R₃ is an acyl group offormula COR₄, in which R₄ is an alkyl group, perfluoroalkyl group, or anaryl substituent containing six to fifteen carbon atoms and having anaromatic ring comprising mono-, di- or tri-substituted aromaticsubstituents activating the aromatic ring; said process comprising thesteps of: (a) hydrolyzing a diastereoisomer of the compound of Formula2:

where: R₁, R₂ and B are as defined above; Y is an oxygen; X is a sulfuror selenium atom, wherein the compound of Formula 2 possesses anabsolute configuration at the P-atom opposite to that desired for theanalogues of Formula 1, said hydrolyzing being done in the presence ofan activator that inverts the absolute configuration of the P-atomresulting in a product of Formula 2, (b) reacting the product of step(a) with a compound of Formula 7:

wherein R₄ is an alkyl, perfluoroalkyl, or a aroyl substituentcontaining six to fifteen carbon atoms, and having an aromatic ringcomprising mono-, di- or tri-substituted aromatic substituentsactivating the aromatic ring; and W is a chlorine, bromine or iodineatom; to yield the compound of Formula 2, where R₁, R₂, B and Z are asdefined above, X is a sulfur or selenium atom, and Y is R₄C(O)O—, inwhich R₄ is as defined above, wherein the compound of Formula 7possesses an absolute configuration at the P-atom opposite to that ofthe diastereoisomer of Formula 2, and identical to that which is desiredfor the analogues of Formula
 1. 15. The process according to claim 14,wherein R_(x) is selected from aroyl, acyl, alkoxycarbonyl,benzenesulfonic, alkyd, trialkylsilyl group and an elongatedoligonucleotide chain.
 16. A process for the synthesis of modifiedP-chiral nucleotide analogues of Formula 1 in the form of a purediastereoisomer possessing a preselected configuration at the P-atom:

wherein: R₁ is a protecting group; R₂ is selected from hydrogen,protected hydroxyl group, vinyl, halogen, nitrile, azide, protectedamine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl,vinyl, ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenylor alkynyl; B is a purine or pyrimidine base; Z is selected from alkyl,aryl, alkenyl, alkynyl, vinyl, ethynyl, aminomethyl or aminoethylsubstituents; R_(x) is a protecting group; Y is an XR₃ substituent,where X is an oxygen, sulfur or selenium atom and R₃ is an acyl group offormula COR₄, in which R₄ is an alkyl group, perfluoroalkyl group, or anaryl substituent containing six to fifteen carbon atoms and having anaromatic ring comprising mono-, di- or tri-substituted aromaticsubstituents activating the aromatic ring; said process comprising thesteps of: (a) hydrolyzing a diastereoisomer of the compound of Formula2:

 wherein: R₁, R₂ and B are as defined above; Y is an oxygen; X is asulfur or selenium atom, wherein the compound of Formula 2 possesses anabsolute configuration at the P-atom opposite to that desired for theanalogues of Formula 1, said hydrolyzing being done in the presence ofan activator that inverts the absolute configuration of the P-atomresulting in a product of Formula 2, (b) reacting the product of step(a) with a compound of Formula 7:

 wherein R₄ is an alkyl, perfluoroalkyl, or a aroyl substituentcontaining six to fifteen carbon atoms, and having an aromatic ringcomprising mono-, di- or tri-substituted aromatic substituentsactivating the aromatic ring; and W is a chlorine; bromine or iodineatom, to yield the compound of Formula 2, where R₁, R₂, B and Z are asdefined above, X is a sulfur or selenium atom, and Y is R₄C(O)O—, inwhich R₄ is as defined above, wherein the compound of Formula 7possesses an absolute configuration at the P-atom opposite to that ofthe diastereoisomer of Formula 2, and identical to that which is desiredfor the analogues of Formula 1; and (c) reacting the product withcompound of Formula 6:

wherein B, R₂ and R_(x) are as defined previously, under anhydrousconditions, in an aprotic organic solvent, in the presence of anactivating reagent, to yield the compound of Formula 1 and isolating thecompound of Formula
 1. 17. The process according to claim 14, whereinthe compound of Formula 2 is obtained by: (i) phosphorylating with aphosphorylating reagent, a substrate of Formula 3:

wherein R₁, R₂ and B are as defined previously; said phosphorylatingreagent comprising a compound of Formula 4:

wherein Z and X are as defined previously, W is a halogen; (ii) followedby hydrolysis without isolation of the intermediate of Formula 5:

where R₁, R₂, B, Z and X are defined previously, and Y is an oxygen atomto yield the compound of Formula
 2. 18. The process according to claim17, further comprising: (i) separating chromatographically the compoundof Formula 2 into two diastereoisomers: and (ii) reacting thediasterosiomer with compound of Formula 7:

wherein: R₄ is an alkyl group, perfluoroalkyl group, or an aroylsubstituent containing six to fifteen carbon atoms and having anaromatic ring comprising mono-, di- or tri-substituted aromaticsubstituents activating the aromatic ring, and W is a halogen.
 19. Theprocess according to claim 14, wherein the activator is an organic baseamine.
 20. The process according to claim 14, wherein the aproticorganic solvent is tetrahydrofuran.
 21. The process according to claim16, wherein the activating reagent is a lithium salt.
 22. A process forthe synthesis of modified P-chiral nucleotide analogues of Formula 1 inthe form of a pure diastereoisomer possessing a preselectedconfiguration at the P-atom:

wherein: R₁ is a protecting group; R₂ is selected from hydrogen,protected hydroxyl group, vinyl, halogen, nitrile, azide, protectedamine group, chloroalkyl, perfluoroalkyl, perfluoroalkoxyl, alkoxyalkyl,vinyl, ethynyl, OQ₁, SQ₁, NHQ₁, where Q₁ stands for alkyl, aryl, alkenylor alkynyl; Y is an oxygen; X is a sulfur or selenium atom wherein thecompound of Formula 2 possesses an absolute configuration at the P-atomopposite to that desired for the analogues of Formula 1, comprising thesteps of: (a) hydrolizing one of two diastereoisomers of formula 2 inthe presence of an activator that inverts the absolute configuration ofthe P-atom resulting in a product of Formula 2

(b) reacting the product of step (a) with a compound of Formula 7:

wherein R₄ is an alkyl, perfluoroalkyl, or a aroyl substituentcontaining six to fifteen carbon atoms, and having an aromatic ringcomprising mono-, di- or tri-substituted aromatic substituentsactivating the aromatic ring; and W is a chlorine, bromine or iodineatom, to yield the compound of Formula 2, where R₁, R₂, B and Z are asdefined above, X is a sulfur or selenium atom, and Y is R₄C(O)O—, inwhich R₄ is as defined above, wherein the compound of Formula 7possesses an absolute configuration at the P-atom opposite to that ofthe diastereoisomer of Formula 2, and identical to that which is desiredfor the analogues of Formula 1; and (c) providing the following compoundof Formula 6:

wherein B, R₂ and R_(x) are as defined previously; (d) reacting thecompound of Formula 6 with the compound of Formula 2 under anhydrousconditions, in an aprotic organic solvent, in the presence of anactivating reagent, to yield compound of Formula 1 of desired absoluteconfiguration at the P-atom; and (e) isolating the compound ofFormula
 1. 23. The process according to claim 14, wherein X is anoxygen, sulfur or selenium atom, and Y of Formula 2 is R₄C(O)O—.